Protein prenylation is a post-translational process where proteins become covalently modified by an isoprenoid group. This modification adds hydrophobicity to the proteins. For all prenylated proteins identified to date, the prenyl group is an absolute requirement for their biological functions. To date, this process has been thoroughly characterised in mammals and yeast with the majority of the studies being performed either in vitro or in tissue-cultured cells. This thesis presents evidence that protein prenylation is a widespread process that is also present in plants. It is focused mainly on in vivo protein prenylation in plants and mammals. In vitro studies of the enzyme farnesyl protein transferase are also discussed.
By using spinach seedlings, whose protein synthesis rates are high, an in vivo method for studying protein prenylation was developed. Spinach, the first plant to be analysed for isoprenoid-modified proteins, was found to contain numerous proteins that were prenylated with metabolites of the isoprenoid precursor mevalonate. The majority of the prenylated proteins originated from cellular organelles. The nature of the covalently-bound isoprenoids was assessed by hydrolysis of lipid-extracted proteins in combination with high performance liquid chromatography (HPLC) analysis. Both thioether-linked farnesyl and geranylgeranyl groups, as well as longer chain isoprenoids, were released.
The soluble fraction of spinach contained an enzyme that could farnesylate the sulphydryl reagent dithiothreitol (DTT). This enzyme was immunoprecipitated with antisera raised against mammalian farnesyl protein transferase (FPT), which strongly suggesting it being a plant FPT. A new assay for measuring prenyl protein transferases was developed. The spinach FPT depended on zinc ions for its activity and functioned preferentially at pH 7.0. DTT inhibited in vivo prenylation of a subset of the prenylated spinach proteins in a dose dependent manner.
Labelling of prenylated proteins in spinach stems was much more intense than that in spinach leaves. In addition, the relative labelling intensity of the prenylated proteins differed markedly in stem and leaf tissue. During greening, the pattern of labelled prenylated proteins was altered. Several chloroplastic proteins displayed an increase in intensity when etiolated seedlings were subjected to light. Etiolated plants contained a higher proportion of thioether-linked farnesyl groups than did plants grown under normal light conditions.
Chloroplasts were shown to contain at least 20 prenylated polypeptides in the first study addressing protein prenylation in this organelle. These were all found in the thylakoid and envelope membrane fractions. A large proportion of the prenylated polypeptides originated from the outer envelope membranes, as assessed by proteolytic treatment of intact chloroplasts. The majority of the chloroplastic prenylated polypeptides were part of multimeric protein complexes, as shown by sucrose density centrifugation of detergent-treated membrane preparations. The thylakoid membranes contained an FPT activity. This is unique as all other prenyl protein transferases characterised to date are soluble enzymes.
Chloroplasts were demonstrated to contain a hitherto undescribed type of prenylated polypeptides. The prenylation of this new type of prenylated polypeptides depended upon expression of chloroplast genes. Furthermore, these polypeptides were modified by isoprenoids via a bond of non-thioether character. The polypeptides encompassing the novel type of modification were readily prenylated when labelling was performed with either mevalonate or farnesol, but not with geranylgeraniol.
Protein prenylation in rats, when studied by mevalonate labelling, was most prominent in the kidney. The majority of the prenylated polypeptides had molecular masses between 21-28 kDa. A substantial proportion of the prenylated proteins in kidney originated from the mitoplast subfraction of mitochondria. In addition to farnesyl and geranylgeranyl groups, which are both well-known prenylating moieties, pentaprenyl and hexaprenyl groups were also bound to rat proteins via thioether linkages, as demonstrated by methyl iodide hydrolysis in combination with HPLC analysis.
Stockholm: Stockholm University , 1998. , 55 p.